With recent technical advances, multiple important signaling pathways that may be the causes of human malignancy have continuously been discovered and dissected. The vast majority of these signaling pathways involve reversible protein phosphorylation, and the information on the location and dynamics of phosphorylation provides important mechanisms on how the signaling networks function and interact. While mass spectrometry has become an exceptionally useful tool for phosphoproteome analyses, extensive experiments are very labor-intensive and cost-prohibitive to most researchers. As a result, despite many large-scale phosphoproteomic studies, the critical need for a routine and effective analysis of relevant phosphoproteins has not been addressed. Through this NIH SBIR Phase I study, we will develop a novel strategy for gel-based phosphoproteome analysis, called Difference Gel Electrophoresis of Phosphoproteome (DiGEP), and translate it into commercial products for simpler phosphorylation discovery assays. The novel design will take advantage of the small molecule platform functionalized with titanium ions for selective binding to phosphoproteins and a UV-based crosslinker to immobilize the reagent onto the bound phosphoprotein. In addition, two different but structurally-similar fluorophores will be used to differentiate the phosphoproteome profiles of two samples ran on a single gel. The strategy will allow quantitative measurements of phosphorylation between the two samples and will pin-point which contrasted phosphoproteins should be further analyzed by mass spectrometry. The proposed approach offers the promise of significant cost reduction and is fully compatible with gel systems and imaging software already developed for Difference Gel Electrophoresis (DIGE).